The Australian saltwater crocodile (Crocodylus porosus) provides evidence that the capacitation of spermatozoa may extend beyond the mammalian lineage

Abstract

Although mammalian spermatozoa only acquire functional maturity as they are conveyed through the male (epididymal maturation) and female (capacitation) reproductive tracts, the degree of post-testicular development necessary to achieve fertilization in other vertebrate species remains far less clear. Indeed, despite reports that the epididymis of birds and reptiles is capable of secreting proteins that bind and modify the sperm surface characteristics, it remains unclear whether capacitation is a pre-requisite for fertilization in these species. Using the ancient reptilian Australian saltwater crocodile as a model, this study was undertaken to explore whether reptile sperm do undergo capacitation-like changes following ejaculation. Our studies revealed that crocodile spermatozoa experienced a rapid and sustained, cyclic-AMP mediated increase in progressive motility following incubation under conditions optimized for the induction of capacitation in mammalian species such as the mouse and human. This response was coupled with elevated levels of phosphorylation associated with both protein kinase A and tyrosine kinase substrates, the latter of which were predominantly localized within the sperm flagellum. In findings that also accord with mammalian spermatozoa, we confirmed a homologue of outer dense fibre 2 as one of the principal substrates for tyrosine phosphorylation. Overall, our findings support the concept that crocodile spermatozoa do undergo a process that is homologous to capacitation in preparation for fertilization of an ovum.

Premammalian origin of the sperm‐specific Slo3 channel

“…Our findings place the Slo3 channel as the first described molecular component putatively involved in sperm function of birds and reptiles. Although it is frequently assumed that sperm capacitation is a process confined to mammals, some studies have reported that nonmammalian species undergo sperm capacitation‐like changes after ejaculation . These observations, in conjunction with our results, suggest that Slo3 channels could mediate a primary K + current essential to acquire fertilizing ability also in the sperm of nonmammalian vertebrates.…”

Post‐ejaculatory modifications to sperm (PEMS)

“…This suggestion is consistent with the term's application by many contemporary reproductive biologists, and underlies the commonly proferred opinion that capacitation is a phenomenon predominantly restricted to mammals (e.g. Nixon et al ., , ). This explicitly restrictive use of ‘capacitation’ will avoid confusion that has arisen by some studies applying the term more generally to PEMS.…”

“…In mammals, the epididymides are specialized for sperm modification, with proteins, glycoproteins and RNA potentially being added, lost and modified and the lipid component of membranes being altered as sperm pass through epididymides of monotremes (e.g. Djakiew & Jones, 1983;Nixon et al, 2011Nixon et al, , 2016b, marsupials (e.g. Temple-Smith & Bedford, 1980) and eutherian mammals (e.g.…”

Modification of Crocodile Spermatozoa Refutes the Tenet That Post-testicular Sperm Maturation Is Restricted To Mammals*

“…Notably, dilution in capacitation medium also enhances the post-thaw survival of cryopreserved crocodile spermatozoa (11). Conversely, crocodile spermatozoa are rendered quiescent upon incubation in bicarbonate-free media formulated to suppress the capacitation of eutherian spermatozoa (10). We contend that such changes may reflect physiological demands imposed by the transferal of sperm storage responsibilities from the male to the female reproductive tract, and the attendant need to alternatively silence and reactivate spermatozoa to enhance their longevity and fertilization competence, respectively.…”

“…Similarly, in the quail it has been shown that most testicular sperm can bind to a perivitelline membrane and acrosome react with no additional advantage being afforded by exposure to capacitation stimuli (9). Although it has been suggested that reptilian spermatozoa also experience minimal post-testicular maturation, this paradigm has recently been challenged by functional analysis of ejaculated spermatozoa from the Australian saltwater crocodile (Crocodylus porosus) (10). In this context, exposure to capacitation stimuli, which elevate intracellular levels of cyclic AMP (cAMP), promoted a significant enhancement of the motility profile recorded in crocodile spermatozoa.…”